Human B cell isolation and culture
B cell isolation
1. Donor blood was obtained with informed consent under guidelines issued by the Medical Ethics Committee.
2. Mononuclear cell fractions were isolated by Ficoll-amidotrizoate centrifugation.
3. B cells were immunomagnetically isolated by positive selection and the CD19 positively selected cells were released from the beads using Detach-a-Bead.
4. Alternatively, B cells were isolated with a Dynal kit based on depletion of non-B cells (negative selection).
5. Yields of positive and negative selections were 98.5 ± 0.5% and 93.8 ± 1.3% pure B cells, respectively, as assessed by staining with a FITC-labeled anti CD19 mAb and flow cytometric analysis of lymphocyte gated cells on a FACSCalibur equipped with CellQuest Software.
B cell culture
1. B cells were isolated by the positive or negative selection methods as described above and were incubated with tetrameric HLA-A2/HPV (100 ng/106 cells) on ice for 40 min, washed, and aseptically sorted for PE staining in a FACSVantage SE (BD Biosciences).
2. Cells with PE staining intensity exceeding channel number 10 were deflected and seeded directly at 1/well in 96-well plates that had been preseeded with EL4.B5 cells (50 Gy irradiated, 5 x 10 4 /well and a T cell supernatant .
3. This T cell supernatant was produced by culturing E-rosette-enriched T cells from a male donor for 36 h in the presence of 5 μg/ml PHA and 10 ng/ml PMA.
4. B cell supernatants (100 μl) were collected at days 9 and 16.
5. All supernatants were analyzed for anti-HLA Ab by CDC and some supernatants for IgG and IgM by ELISA.
6. Some supernatants were tested for binding to streptavidin.
Reference
1. Wen, L., M. Hanvanich, C. Werner-Favre, N. Brouwers, LH Perrin, RH Zubler. Limiting dilution assay for human B cells based on their activation by mutant EL4 thymoma cells: total and antimalaria responder B cell Frequencies. Eur J Immunol. 1987; 17: 887
2. Arend Mulder, Chantal Eijsink, Marrie J. Kardol, Marry EI Franke-van Dijk, Sjoerd H. van der Burg, Michel Kester, Ilias IN Doxiadis, and Frans HJ Claas. Identification, Isolation, and Culture of HLA-A2- Specific B Lymphocytes Using MHC Class I Tetramers. J Immunol. 2003; 171: 6599.
The P10 two-color display module all uses the JX5020 driver chip from Taiwan Macroaccumulation. The LEDs of each branch are correspondingly driven by an IC pin. One IC pin drives one LED. We use static cross-current The driving method ensures that the current of each IC can be guaranteed, and the normal guarantee will not cause the entire module to heat up due to the different scanning methods, which will cause damage to the IC, and the brightness of the LED will also be similar to the static cross current. The static cross-current drive ensures the stability of the entire screen.
When all the LED display modules are tested, paste QC, pass, and then perform the first semi-finished product test aging. The longer the aging time, the better, let him exceed 24 hours as much as possible, and then we will display the display module Put on the bottom shell, ready to start pouring.
The glue is filled with a fully automatic glue filling machine. After the ab component glue is combined, the filling is carried out according to the ratio of 10:1. It is better not to lower or increase the ratio. The ratio of 10:1 is just right. , It can be surface-dried in about two hours, and it will dry out in about 4-6 hours. Such glue will have a good guarantee for long-term use.
After the module is filled with glue, place the module on a horizontal plane, and test with a level meter, and then try to make each module as flat as possible, complete the 4 corners of the horizontal position on the same level, the choice of AB glue It is also very important. The AB glue choice must use more than 40 yuan per kilogram of glue. Its adhesion and durability will be very good. It cannot be filled with shoddy glue. It will crack after a long time. The screen will leak. Then put all the display modules in their masks and arrange them to start packaging.
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